D I S C U S S I O N
H. Leclerc, France. Formal discussion.
Research into the identification of coliform bacteria (colimetry) is characterised by the great
diversity of the techniques proposed. Some make use of filtration through membranes and various
culture media. Others depend upon liquid culture media and the statistical interpretation of the results
(MPN method).
However, most authors have one preoccupation - whatever the method they use, they try to obtain
evidence in great detail concerning the specificity of the contamination by the precise identification of
species of faecal origin, which some authors would like to place under the group of faecal coliforms.
The species Escherichia coli is certainly the most important of these, where it is necessary to
differentiate it from other coliforms by very simple methods. In liquid media one may use as a basis of
selection the temperature of incubation (generally 44°C) or on the other hand growth inhibitors
(Eijkman's or Mackenzie's tests, formate, lactose, bile salt media, etc.).
Membrane filtration, though the results may be open to differing interpretation, have the
advantage of speed over those using liquid media. They are also performed in different ways -
incubation at 44°C which allows only E.coli to develop; at 37°C on the other hand, all the coliform
organisms grow but also, unfortunately, certain saprophytic bacteria which we have reported
l
.
These high temperatures, applied directly to samples of filtered water may undoubtedly prevent
the growth of certain cells belonging to the species under investigation. This has prompted the author
to propose their new techniques which depends upon * growth of E.coli at its optimum growth, i.e.
37°C and
2 identification of the E.coli by lysis with bacteriophage.
The filtration of samples having been made through two membranes, one is incubated directly at
37°C and the total number of coliform organisms is allowed to grow, while the other is subjected to a
concentrated extract of anti B.coli bacteriophage, which inhibits their growth but allows the coliforms
other than E.coli to grow. According to the authors this method would be particularly useful for
detecting slight contaminations that would escape detection under the unfavourable influence of
higher temperatures. In fact, this replaced the 44°C temperature test by a phage test.
This original proposal deserves considerations for the following reasons:—
1. It is generally admitted that the surest way of species identification is that which rests on a
biochemical test and ultimately on an antigenic reaction
2 . It is in fact only an extreme
simplification and in making use of a single test (temperature, for instance) one may incur errors,
the importance and frequency of which should be known. Under these conditions it would be
advisable to know exactly the nature of the specificity of the bacteriophages used by the authors
and to compare it with the high temperature test. The Enterobacteriaceae and especially the
coliforms possess several common antigens and it is not unusual for bacteriophages to act on one or
the other.
2. The inhibitory nature of the techniques introduced for the detection of E.coli (high temperature,
bacteriocides etc.) is a disadvantage. But is it more so than the natural antagonistic action which
may exist between species capable of a growth at 37°C? This is equivalent to saying that the
proposed method could be applicable in the case of low contamination. For heavier pollution it
would be advisable that it is controlled. In this case would the observer be able to distinguish
between the two cultures when the two membranes are well covered with colonies? Such questions
do not detract from the originality of the authors' proposals which need to be explored and tested
in order to define their significance more precisely.
REFERENCES
1 LECLERC H. et BUTTIAUX R. Frequence des Aeromonas dans les eaux d'alimentation Ann. Inst.
Pasteur, 1962,103 97-100.
2 LECLERC H. La reaction d'augmentation du titre bacteriophagique Ann. Inst. Pasteur Lille; 1963,
XIV 145-158.
K. Grunnet, Denmark.
Is it possible that you have not identified the colonies that you presume to be E.coli in the
comparison of counts at 37° and 44° or that your Imvic tests were done at 37°C? If so is it not
possible that you might have included a group of coliforms?
Reply
Typical blue colonies were picked and transferred to slants Im VIC tests were performed and all
typical blue colonies grown on MmFc were found to be of the type ++ -. Hundreds of such isolated
colonies were tested without mistake. As mentioned in the paper, of all Im VIC identified strains
93.5% could be identified by specific E. Coliphages.
83
H. Leclerc, France. Formal discussion.
Research into the identification of coliform bacteria (colimetry) is characterised by the great
diversity of the techniques proposed. Some make use of filtration through membranes and various
culture media. Others depend upon liquid culture media and the statistical interpretation of the results
(MPN method).
However, most authors have one preoccupation - whatever the method they use, they try to obtain
evidence in great detail concerning the specificity of the contamination by the precise identification of
species of faecal origin, which some authors would like to place under the group of faecal coliforms.
The species Escherichia coli is certainly the most important of these, where it is necessary to
differentiate it from other coliforms by very simple methods. In liquid media one may use as a basis of
selection the temperature of incubation (generally 44°C) or on the other hand growth inhibitors
(Eijkman's or Mackenzie's tests, formate, lactose, bile salt media, etc.).
Membrane filtration, though the results may be open to differing interpretation, have the
advantage of speed over those using liquid media. They are also performed in different ways -
incubation at 44°C which allows only E.coli to develop; at 37°C on the other hand, all the coliform
organisms grow but also, unfortunately, certain saprophytic bacteria which we have reported
l
.
These high temperatures, applied directly to samples of filtered water may undoubtedly prevent
the growth of certain cells belonging to the species under investigation. This has prompted the author
to propose their new techniques which depends upon * growth of E.coli at its optimum growth, i.e.
37°C and
2 identification of the E.coli by lysis with bacteriophage.
The filtration of samples having been made through two membranes, one is incubated directly at
37°C and the total number of coliform organisms is allowed to grow, while the other is subjected to a
concentrated extract of anti B.coli bacteriophage, which inhibits their growth but allows the coliforms
other than E.coli to grow. According to the authors this method would be particularly useful for
detecting slight contaminations that would escape detection under the unfavourable influence of
higher temperatures. In fact, this replaced the 44°C temperature test by a phage test.
This original proposal deserves considerations for the following reasons:—
1. It is generally admitted that the surest way of species identification is that which rests on a
biochemical test and ultimately on an antigenic reaction
2 . It is in fact only an extreme
simplification and in making use of a single test (temperature, for instance) one may incur errors,
the importance and frequency of which should be known. Under these conditions it would be
advisable to know exactly the nature of the specificity of the bacteriophages used by the authors
and to compare it with the high temperature test. The Enterobacteriaceae and especially the
coliforms possess several common antigens and it is not unusual for bacteriophages to act on one or
the other.
2. The inhibitory nature of the techniques introduced for the detection of E.coli (high temperature,
bacteriocides etc.) is a disadvantage. But is it more so than the natural antagonistic action which
may exist between species capable of a growth at 37°C? This is equivalent to saying that the
proposed method could be applicable in the case of low contamination. For heavier pollution it
would be advisable that it is controlled. In this case would the observer be able to distinguish
between the two cultures when the two membranes are well covered with colonies? Such questions
do not detract from the originality of the authors' proposals which need to be explored and tested
in order to define their significance more precisely.
REFERENCES
1 LECLERC H. et BUTTIAUX R. Frequence des Aeromonas dans les eaux d'alimentation Ann. Inst.
Pasteur, 1962,103 97-100.
2 LECLERC H. La reaction d'augmentation du titre bacteriophagique Ann. Inst. Pasteur Lille; 1963,
XIV 145-158.
K. Grunnet, Denmark.
Is it possible that you have not identified the colonies that you presume to be E.coli in the
comparison of counts at 37° and 44° or that your Imvic tests were done at 37°C? If so is it not
possible that you might have included a group of coliforms?
Reply
Typical blue colonies were picked and transferred to slants Im VIC tests were performed and all
typical blue colonies grown on MmFc were found to be of the type ++ -. Hundreds of such isolated
colonies were tested without mistake. As mentioned in the paper, of all Im VIC identified strains
93.5% could be identified by specific E. Coliphages.
83